This project is aimed at understanding the mechanism whereby alternative pre-mRNA processing is used to generate tissue-specific RNAs coding for different proteins. The gene under study is that coding for the peptide hormones calcitonin (CT) and calcitonin-gene-related peptide (CGRP). The two peptides are produced from a single pre-mRNA to include an alternative 3' terminal exon, exon 4, to produce CT or exclude exon 4 to produce CGRP. Calcitonin and mRNA including exon 4 are produced mainly in thyroid C cells; CGRP and mRNA excluding exon 4 are produced mainly in neuronal cells. Malignant thyroid C cells and small lung cell carcinomas express both RNAs and peptides, presumably due to a loss of processing regulation. We have identified an intriguing intron enhancer that regulates this processing choice by altering polyadenylation cleavage efficiency of the alternative 3'-terminal exon. This enhancer is located downstream of the regulated exon and is the only known distal tissue-specific enhancer known to regulate polyadenylation. In previous work we have shown that the enhancer has a CORE resembling a pseudo exon and binds splicing factors, including U1 snRNPs, SR proteins (ASF/SF2 and SRp20), and PTB. In addition, we have shown that the enhancer contains additional sequences important for both inclusion and exclusion of exon 4 that bind unknown factors that may be important for exon skipping in neuronal and malignant cells. This proposal contains experiments to further characterize the enhancer in both normal and malignant cells. Experiments are directed at the mechanism whereby the enhancer facilitates polyadenylation and exon inclusion in most cells, or exon exclusion in neuronal and malignant cells. In addition, the proposal contains experiment directed at the role pseudo exon intron elements might play in more generalized splicing events. Specific aims include: 1) Identify new factors involved in enhancer function, 2) Determine the role of these factors in tissue-specific inclusion and exclusion of exon 4; and 3) Determine the role of the enhancer in general and regulated splicing. The overall goal of these experiments is to understand how an enhancer resembling an exon participates in both polyadenylation and splicing of neighboring exons.